This invention relates to the in vitro production of human angiogenic factor from human foreskin fibroblast cell lines.
Angiogenesis, or the ability to stimulate blood vessel growth, is important to burn and wound healing and certain inflammatory reactions as well as to tumor growth. The substance that is released by tumors and provides vascularization has been named tumor angiogenesis factor (TAF) by Dr. Judah Folkman of the Harvard Medical School. J. Exptl. Med. 133, 275-88 (1971); Ann. Surg. 175, 409-16 (1972); Cancer Res. 34, 2109-13 (1974); and Advan. Cancer Res. 19, 331-58 (1974). Provision for the availability of TAF is particularly useful as an aid to the search for ways to inhibit neovascularization. While TAF also finds use in the development of tests such as an angiogenic assay or a diagnositc screening test for neoplasia, for use in patient treatment an angiogenic material derived from normal rather than tumor cells would be much preferred from the standpoint of safety.
Various investigators in the field have reported heretofore that angiogenic activity is either absent or weak in most normal tissue extracts or grafts or cell culture extracts. Thus, in a paper by Folkman on "Tumor Angiogenesis", Advan. Cancer Res. 19, 331-58 (1974), only two exceptions were found to the "general rule that normal adult and embryonic tissues do not induce neovascularization." The observed exceptions were that pieces of embryonic and adult mouse kidney induced mild neovascularization when grafted to the chorioallantoic membrane (CAM) of the chick embryo and that salivary gland from the adult mouse also seemed to be able to induce neovascularization on the CAM. In another group from the same research group by Auerback et al., Int. J. Cancer 15, 241-5 (1975), it was reported that control or irradiated placental and muscle tissue from rabbits did not cause a vascular response comparable to that obtained with Walker rat carcinosarcoma and other tumors either with or without irradiation. Klogsbrun et al., Cancer Res. 36, 110-14 (1976), subsequently reported that TAF was not detected in normal liver and kidney or in density-inhibited BALB/c primary mouse embryo or early passage human skin fibroblasts, but that density-inhibited BALB/c 3T3 and WI 38 human embryonic lung fibroblasts did produce TAF.
Another group of investigators, Wolf and Hubler, Arch Dermatol 111, 321-27 (1975), also investigated the elaboration of TAF by various implated tumors. In parallel tests, angiogenesis was found to be conspicuously absent after implantation of control materials and nevoid or normal cutaneous components with the exception of human and hamster epidermis.
Gimbrone and Gullino, J. Nat. Cancer Inst. 56 (2), 305-18 (1976), studied the implantation of mouse mammary tissue in rabbit cornea. They found that while neoplastic tissue stimulated blood vessel growth, normal tissue rarely produced any vascular change.
Phillips et al., Int. J. Cancer 17, 549-58 (1976) and 23, 82-88 (1979), found that rat liver, normal human kidney and various other normal or foetal tissues failed to induce angiogenesis.
On the other hand, significant angiogenic factor has been found in normal tissue by several investigators. Thus, Wolf et al., J. Invest. Dermatol. 61, 130-41 (1973), reported on an epidermal angiogenic factor from separated hamster epidermis and epidermal extracts as an exception to the general finding that normal tissues do not induce neovascularization when implanted onto the hamster cheek pouch.
Huseby et at., Microvas. Res. 10, 396-413 (1975), reported on a normal tissue explant system for 1-day old mouse testes that stimulated proliferation of host blood vessels in adult mice.
The corpus luteum was also found to produce a diffusible substance similar to TAF as a blood growth stimulant by Oehme et al., East German Patent 128,368 (1977); and by Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 75 (2), 847-851 (1978).
All of the foregoing normal human cells are derived from fresh tissue or primary cultures except the WI 38 cells. Fresh tissue and primary cultures are not, however, generally suitable sources of angiogenic factor except for limited research purposes or small scale production. In order to provide a commercially significant source of angiogenic factor in terms of ready availability and adequate supply, production by cell culture of a suitable cell line is deemed necessary. As a practical matter, the cell line should not only have specific angiogenic factor activity, but it should also have good cell growth characteristics is terms of rapid growth and production of angiogenic factor on a sustained basis.
The term "cell line" is used herein in conformance with the well-known art accepted definition published by Federoff in the Tissue Culture Association Manual, Vol. 1, No. 1, pp. 53-57 (1975).
Further background information on angiogenic activity in normal tissues can be had by reference to a review by Ausprunk in Chapter 10 of "Handbook of Inflammation", Volume 1, at pages 321 and 343-346, Series ed. Glynn et al., Elsevier/North-Holland Biomedical Press, 1979.